Cystic fibrosis (CF) is a systemic disorder that results when mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane glycoprotein, lead to a reduction in apical membrane chloride transport. CFTR is a cAMP-dependent chloride channel that regulates fluid composition in the respiratory and gastrointestinal tracts. CF is a heritable disease that follows an autosomal recessive pattern of transmission. It is the most common invariably lethal genetic disease in the United States, with frequency among Caucasians being one in two thousand. One in twenty are carriers of the defective gene. CF is characterized by abnormal endocrine and exocrine gland function. In CF. unusually thick mucus leads chronic pulmonary disease and respiratory infections, insufficient pancreatic and digestive function, and abnormally concentrated sweat. Seventy percent of the mutant CFTR alleles in the Caucasian population result from deletion of phenylalanine at position 508 (ΔF508-CFTR), the result of a three base pair deletion in the genetic code. Other mutations have also been described and many may exist. The ΔF508-CFTR mutation results in a CFTR protein capable of conducting chloride, but absent from the plasma membrane because of aberrant intracellular processing. Under usual conditions (37° C.), the ΔF508-CFTR protein is retained in the endoplasmic reticulum (ER), by prolonged association with the ER chaperones, including calnexin and hsp70. The retained CFTR protein is then targeted for degradation by the ubiquitin proteasome pathway. Over expression of ΔF508-CFTR can result in ΔF508-CFTR protein appearing at the cell surface, and this protein is functional once it reaches the cell surface. The ΔF508 “trafficking” block is also reversible by incubation of cultured CF epithelial cells at reduced temperatures (25-27° C.). Lowered temperature results in the appearance of CFTR protein and channel activity at the cell surface, suggesting an intrinsic thermodynamic instability in ΔF508-CFTR at 37° C. that leads to recognition of the mutant protein by the ER quality control mechanism, prevents further trafficking, and results in protein degradation. High concentrations of glycerol (1 M or 10%), a protein stabilizing agent or chemical chaperone, also appears to facilitate movement of ΔF508-CFTR from the ER to the plasma membrane.
Some of the palliative treatments involve the administration of biologically active proteins or chemical compounds to decrease the viscosity of secretions, or to suppress chronic infections of the airways. These treatments have a number of limitations, and do not address the illness directly, but rather attempt to treat the symptoms. Some require continuous use at fairly high doses while others have short effective half-lives. Tolerance to the active ingredient often develops rendering the composition functionally useless. In addition to problems associated with tolerance, the substances themselves or their metabolic by-products or carriers can quickly reach toxic levels in the patient's system which impair kidney or liver function. Further, the chemical compounds themselves can be rapidly destroyed by catabolic enzymes, found in the cells and serum such as aminases, oxidases and hydrolases. Many of these enzymes are also found in hepatic cells, the principal sites for cleansing of the blood. Those able to survive cellular and hepatic catabolic processes are quickly eliminated from the patient's system by the kidneys. Consequently, in vivo retention times for active compounds are extremely short and the ability to achieve any sort of sustained biological effect becomes nearly impossible or, at least, impractical.
Gene therapy for cystic fibrosis has been attempted, but has not been successful to date for a number of reasons, including problems with delivery of the gene to airway cells, insufficient levels of gene expression, inadequate duration of gene expression, and toxicity of the gene therapy preparations.
A recent publication used 4-phenylbutyrate (4PBA) to enable a greater fraction of ΔF508-CFTR to escape degradation and appear at the cell surface (Rubenstein, R. C., Egan, M. E., and Zeitlin, P. L. In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenyl butyrate in cystic fibrosis epithelial cells containing delta-F508-CFTR. J. Clin. Invest. 100:2457-65, 1997). Briefly, primary cultures of nasal polyp epithelia from CF patients (ΔF508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (ΔF508/W1282X) were exposed to 4PBA for up to 7 days in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM 4PBA. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR, consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of ΔF508-CFTR.
As 4PBA is an analogue of butyrate, a known transcriptional regulator of CFTR expression (Cheng, S. H., Fang, S. L., Zabner, J., Marshall, J., Piraino, S., Schiavi, S. C., Jefferson, D. M., Welsh, M. J., and Smith, A. E. Functional activation of the cystic fibrosis trafficking mutant ΔF508-CFTR by expression. Am. J. Physiol. 268:L615-24, 1995), it was hypothesized that 4PBA might increase transcription of the ΔF508-CFTR allele (Rubenstein et al.). If it were a transcriptional regulator, 4PBA might thereby increase levels of ΔF508-CFTR protein, and by mass action, would force some ΔF508-CFTR to bypass quality control in the ER. Such a mechanism would be consistent with the observations that butyrate itself can induce cAMP-responsive chloride secretion in a ΔF508-homozygous pancreatic acinar cell line (Cheng et al.). The results observed were consistent with 4PBA increasing the amount of ΔF508-CFTR protein produced, but their data demonstrated that this was not due to a transcriptional regulatory effect of 4PBA on the CFTR gene. In immunoblot experiments, increased CFTR immunoreactivity was observed in the 4PBA-treated samples. Increased CFTR immunoreactivity was also observed by immunocytochemistry after 4PBA treatment, but no changes in CFTF RNA levels were found with 4PBA treatment. The authors further stated that butyrate and 4PBA have effects in 1133-1 cells that are qualitatively different from one another. Respiratory epithelial cells treated with 1-2 mM 4PBA are healthy, grow at a similar rate and with a similar morphology to control cells, and express CFTR channel activity at the plasma membrane. Equimolar concentrations of butyrate caused morphologic changes in IB3-1 cells, with rounding of cells and decreased growth rate.
This seems to indicate that 4PBA and butyrate may have different toxicity profiles and dose-response relationships. In addition, other published observations with butyrate in ΔF508-CFTR transfected C-127 cells found that the ˜180-kD mature glycosylated species of CFTR was not observed after 5 mM butyrate treatment for 24 hours, despite a massive increase in ΔF508-CFTR mRNA as demonstrated by Northern analysis (Cheng et al.). This data thus did not demonstrate any effects of butyrate on CFTR protein levels or function, only changes in cellular morphology and cell death (Rubenstein et al.). Rubenstein et al observed no increases in CFTR mRNA in response to 4PBA and indicated that the mechanism of action of 4PBA was not similar to that of butyrate or related to increasing ΔF508-CFTR transcription. In addition, no increases in cAMP-stimulation was observed which would be indicative of chloride ion transport even after treatment with up to 300 mM butyrate (Cheng et al.).
These data argue against any beneficial or therapeutic effect of butyrate on cystic fibrosis. In fact, some authors even stated that butyrate is likely too toxic to use clinically (Rubenstein et al.). Further, the authors made a strong case that 4PBA, which was indicated to be possibly clinically useful, works though a mechanism, which although unknown, is different from butyrate. Taken together, the use of butyrate, and the newer butyrate-derived compounds claimed, as CF therapeutics is contra-indicated according to these reports. Moreover, 4PBA has been used in a few CF patients clinically, but was not well tolerated due to large number of pills required (i.e. very short half-life), and other side effects and, in consideration, that study was terminated.